JChemEcol DOI 10.1007/s10886-016-0800-1

Evidence that Cerambycid Beetles Mimic Vespid Wasps in Odor as well as Appearance

Robert F. Mitchell1,2 & Tomislav Curkovic3 & Judith A. Mongold-Diers1 & Lara Neuteboom4 & Hans-Martin Galbrecht4 & Armin Tröger4 & Jan Bergmann5 & Wittko Francke4 & Lawrence M. Hanks1

Received: 13 June 2016 /Revised: 20 September 2016 /Accepted: 30 November 2016 # Springer Science+Business Media New York 2016

Abstract We present evidence that cerambycid species that are dominant compound. Females of M. robiniae produced six dif- supposed mimics of vespid wasps also mimic their model’s odor ferent spiroacetals, one of which was not produced by M. caryae, by producing spiroacetals, common constituents of vespid alarm (2E,7E)-2-ethyl-7-methyl-1,6-dioxaspiro[4.5]decane, and five pheromones. Adults of the North American cerambycids that were shared with M. caryae, including the dominant Megacyllene caryae (Gahan) and Megacyllene robiniae (2E,8E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane. The latter (Forster) are conspicuously patterned yellow and black, and are compound is the sole spiroacetal produced by both males and believed to be mimics of aculeate Hymenoptera, such as species females of a South American cerambycid species, Callisphyris of Vespula and Polistes. Adult males of M. caryae produce an apicicornis (Fairmaire & Germain), which is also thought to be a aggregation-sex pheromone, but both sexes produce a pungent wasp mimic. Preliminary work also identified spiroacetals of odor when handled, which has been assumed to be a defensive similar or identical structure released by vespid wasps that co- response. Headspace aerations of agitated females of M. caryae occur with the Megacyllene species. contained 16 compounds with mass spectra characteristic of spiroacetals of eight distinct chemical structures, with the dom- Keywords Spiroacetal . chemical defense . chemical inant compound being (7E,2E)-7-ethyl-2-methyl-1,6- mimicry . Cerambycidae . Megacyllene caryae . Megacyllene dioxaspiro[4.5]decane. Headspace samples of agitated males of robiniae . Callisphyris apicicornis M. caryae contained five of the same components, with the same

Electronic supplementary material The online version of this article (doi:10.1007/s10886-016-0800-1) contains supplementary material, Introduction which is available to authorized users. Batesian mimics are species that have gained protection from * Robert F. Mitchell natural enemies by resembling other species that are unpalat- [email protected] able or otherwise defended (Ruxton et al. 2004). Many species of beetles in the Cerambycidae appear to be Batesian mimics 1 Department of Entomology, University of Illinois at of other insects, including tenebrionid beetles (Raske 1967; Urbana-Champaign, Urbana, IL 61801, USA Slobodchikoff 1987), ants (Berlocher et al. 1992), and even 2 Present address: Department of Biology, University of Wisconsin spiders and true bugs (Linsley 1959). Mimicry of lycid beetles Oshkosh, 142 Halsey Science Center, 800 Algoma Blvd., is well documented, and predatory cerambycids may mimic Oshkosh, WI 54901, USA their lycid prey (Eisner et al. 2008) or emit similar warning 3 Departamento de Sanidad Vegetal, Universidad de Chile, PO Box odors (Dettner 1987). Mimicry of wasps also is particularly 1004, Santiago, Chile common among diurnal cerambycids that feed on pollen, and 4 Institut für Organische Chemie, Universität Hamburg, such vespiform mimics have been described from several gen- 20146 Hamburg, Germany era in the cerambycid subfamilies Cerambycinae, Lepturinae, 5 Instituto de Química, Pontificia Universidad Católica de Valparaíso, and Necydalinae (Švácha and Lawrence 2014). In fact, some Valparaíso, Chile of these species so closely resemble wasps in color pattern, JChemEcol morphology, and walking and flight behavior that human ob- C. apicicornis was conducted independently, and was includ- servers may be deceived (Linsley 1959). ed here because this species is similar to the Megacyllene Closer scrutiny of mimicry systems has revealed recently that species in the chemistry of its defensive response and, of mimicry extends beyond visual cues, with mimics also resem- course, in its mimicry of aculeate Hymenoptera. bling their models in production of sound (Barbero et al. 2009) and odor (Vereecken and McNeil 2010). Here, we present evi- dence that cerambycid species that mimic hymenopterans also Methods and Materials resemble their model’s odor by producing spiroacetals, common constituents of alarm pheromones released from venom glands Sources of Insects Adults of M. caryae were captured alive of the models (Francke and Kitching 2001). Our study species with cross-vane panel traps (black corrugated plastic, are the congeners Megacyllene caryae (Gahan) and M. robiniae AlphaScents, Portland, OR, USA; see Graham et al. 2010) dur- (Forster) (Cerambycinae), native to eastern North America, and ing May 2011 at Forest Glen Preserve (Vermilion County, IL, Callisphyris apicicornis (Fairmaire & Germain) (Necydalinae), USA; 40.015°, −87.568°). Trap lures were polyethylene reseal- native to Chile and Argentina (Bezark and Monné 2013). able sachets containing 50 mg of citral (Sigma-Aldrich, St. Adult M. caryae and M. robiniae are similar in appearance, Louis, MO, USA) in 1 ml isopropanol. Citral is an isomeric conspicuously patterned yellow and black, and are believed to blend of neral and geranial, both pheromone components of be mimics of aculeate hymenoptera, as are many other mem- M. caryae (Lacey et al. 2008), and attracts adults of both sexes bers of the cerambycine tribe Clytini (Linsley 1959). Adults of (e.g., Handley et al. 2015). Adults of M. robiniae were collected the two species are active at opposite ends of the season, with by hand from inflorescences of Solidago species on roadsides M. caryae flying in early spring and M. robiniae flying in near Mazonia State Fish and Wildlife Area (Grundy County, IL, early autumn (Dusham 1921). Similar temporal allopatry has USA; 41.197°, −88.269°) during September 2011. Conspecific been reported for syrphid fly mimics of Hymenoptera, and is adults of the Megacyllene species were housed together in the thought to minimize contact with insectivorous birds during laboratory in aluminum screen cages under ambient conditions the summer nesting season, when fledglings have not yet (~12:12 h L:D, ~20 °C), and provided with 10% aqueous su- learned to avoid the vespid models (Waldbauer 1988). crose solution as nourishment (glass vial with cotton wick). Adults of both Megacyllene species are diurnal and feed on Adults of M. robiniae also were provided freshly cut inflores- pollen: adults of M. caryae visit flowers of Crataegus and cences of Solidago species. Quercus species (Dusham 1921; LMH unpubl. data), whereas Adults of C. apicicornis were reared from branches of larval adults of M. robiniae visit flowers of Solidago species host plants (primarily quince, Cydonia oblonga Miller, (Wheeler et al. 1988). The diurnal adults of C. apicicornis also Rosaceae), collected at several locations in the vicinity of are frequently found on flowers (Curkovic and Ferrera 2012) Santiago, Chile (BLo Cañas^, −33.533°, −70.557°; BEl Olivar^, and, like adults of closely related species, are thought to mimic −34.232°, −70.874°) during August–September of 2007 and hymenopterans, such as species of Hypodynerus (Vespidae) 2008. Branches were placed in Flanders cages (Curkovic and and Pepsis (Pompilidae), resembling them in color, form, and Ferrera 2012) in the laboratory (~20 ± 5 °C, natural photoperiod). flight behavior (Barriga and Peña 1994). Cages were checked every morning, and freshly emerged adults Males of M. caryae emit an aggregation-sex pheromone were collected and sexed (males are smaller than females, and (sensu Cardé 2014) composed of nine components, including have longer antennae). Adults emerged from September– isomers of 2,3-hexanediol and 2-methylbutan-1-ol (common December and were used in experiments 1–13 d after emergence. pheromone components among cerambycid species in the Beetles were held in individual 500 ml plastic vials, and provided subfamily Cerambycinae; Millar and Hanks 2016), but also with 5% aqueous sugar solution as nourishment. including compounds that are better known as floral and wood volatiles: (S)-(− )-limonene, 2-phenylethanol, (−)-α-terpineol, Collection and Analysis of Volatiles Solid phase nerol, neral, and geranial (Lacey et al. 2008; Mitchell et al. microextraction (SPME) was used to collect volatiles produced 2012). Adult M. robiniae of both sexes produce a secretion, by individual beetles of the two Megacyllene species. Beetles emanating from metasternal glands, when they are handled, were placed in 50 ml glass flasks that were capped with alumi- which is composed primarily of 2-(1,3-hexadien-1-yl)-5- num foil, and agitated by vigorously shaking the flasks for a few methyltetrahydrofuran (Wheeler et al. 1988). seconds. A SPME fiber (100 μm polydimethylsiloxane, Supelco During research on M. caryae (Mitchell 2012), it was dis- Inc., Bellefonte, PA, USA) was inserted through the foil and covered that adults of both sexes emit a pungent odor when exposed to headspace volatiles for 20 min. Headspace volatiles handled, which was assumed to be a defensive response. The were analyzed from ten beetles of each sex of M. caryae,and research reported here was initially intended to characterize five females of M. robiniae. Trace compounds identified in these the chemical nature of this defensive response, as well as that initial samples were later isolated by collecting volatiles in this of a similar response by adults of M. robiniae. Research on same manner, but from groups of 4–5 agitated beetles. JChemEcol

SPME fibers were desorbed in the injection port of a Hewlett- Source of Reference Compounds Standards of spiroacetals Packard 6890 gas chromatograph coupled to a 5973 mass selec- were not commercially available, so racemates of diastereo- tive detector (Hewlett Packard, Palo Alto, CA, USA). The GC meric mixtures of structures A-G (Booth et al. 2006;Francke was fitted with an AT-5 ms column (30 m, 0.25 mm i.d., 0.25 μm and Kitching 2001) were prepared according to established film; Alltech Associates, Inc., Deerfield, IL, USA), the injector methods (Doubský et al. 2004;Jacobsonetal. 1982; Phillips temperature was 250 °C, and the column oven programmed et al. 1980). Following the approach of Jacobson et al. (1982), from 40 °C (1 min. hold) to 160 °C at 10 °C/min−1. Helium the syntheses of the new 2-methyl-7-propyl-1,6- was the carrier gas, and mass spectra were acquired at 70 eV. dioxaspiro[4.6]undecane (structure H) and 8-propyl-1,7- Chemical structures of compounds were tentatively assigned dioxaspiro[5.6]dodecane (structure I) are described in Online according to their mass spectra using a library (National Resource 1. Institute of Standards and Technology, Gaithersburg, MD, USA) or by comparison with published spectra (Francke and Structural Features and Identification of Spiroacetals Kitching 2001). Structure assignments were unambiguously Intramolecular ring closure of a straight chain ketone, with confirmed by comparison of retention times and mass spectra stereochemically non-defined secondary hydroxy groups at either with those of authentic reference samples. Peak areas were cal- side of the carbonyl group, yielded a mixture of bicyclic acetals culated as percentages relative to the dominant spiroacetal struc- called spiroacetals (synon. spiroketals). The two rings of the sys- ture, and percentages of each compound were averaged across tem share a new stereogenic carbon, referred to as the spirocenter. samples within a species and according to sex. In the most widespread spiroacetal system, the 1,6- Volatiles from C. apicicornis were collected by placing in- dioxaspiro[4.5]decanes, a six-membered ring (tetrahydropyran) dividual males and females in cylindrical glass chambers (4 cm and a five-membered ring (tetrahydrofuran) share the spirocenter diam. × 30 cm long) connected by Teflon® tubing to an adsor- (Fig. 1A). In the other frequently found system, the 1,7- bent collector (~150 mg activated charcoal; Fisher Scientific, dioxaspiro[5.5]undecanes, two tetrahydropyran substructures Waltham, MA, USA). Beetles were allowed 5 min to acclimate are linked (Fig. 1B). When the substituent adjacent to the oxygen before charcoal-purified air was pulled through the apparatus at of the first ring is positioned at the opposite side to the oxygen of − 1 l/min 1 for 2–4 h. Beetles were aerated between 1000 and the second ring, it is assigned to keep the more stable BE- 1400 h (when adults were most active in the field), under the configuration^, while the alternative represents the less stable same environmental conditions as used in rearing adult beetles BZ-configuration^. An alkyl substituent adjacent to the oxygen (see above), but under fluorescent lighting. Collectors were in the tetrahydropyran substructure of a spiroacetal keeps the eluted with ~1.5 ml hexane (SupraSolv, Merck, Darmstadt, equatorial position (Deslongchamps et al. 1981; Francke et al. Germany), and extracts were concentrated to 5 μl under a gentle 1980), whereas the stabilizing anomeric effect (Perron and nitrogen stream. Samples (1 μl) were injected into a GC17A Albizati 1989) directs the oxygen of the alternate ring to the axial gas chromatograph coupled to a GCMSQP5050A mass spec- position causing (E)-configuration. The thermodynamical stabil- trometer (Shimadzu, Kyoto, Japan). The GC was equipped with ity and polarity of E/Z-isomers are different (Booth et al. 2009; a 30 m × 0.25 mm VF5-ms fused silica capillary column Pothier et al. 1992), and they separate readily by gas chromatog- (Agilent, Santa Clara, CA, USA), the injector temperature raphy. The (E,E)-isomers elute first (Francke et al. 1981;Heiduk was set to 250 °C, and the oven programmed from 50 to et al. 2015; Kitching et al. 1989), because the oxygens are more − 270 °C at 8 °C/min 1. Helium was the carrier gas, and the mass shielded by the substituents, which renders these stereoisomers spectra were acquired at 70 eV. Structures of compounds were less polar. The stereoisomers showing (Z,Z)-configuration confirmed by co-injection of the natural extract with authentic (Fig. 1) elute last. The two oxygens, keeping equatorial positions, reference samples. are even less shielded. These more polar compounds are unstable

Fig. 1 A 2,9-Dihydroxyundecan-5-one forms 4 pairs of enantiomers of 7-ethyl-2-methyl-1,6-dioxaspiro[4.5]decane (Structure E). Only one enantiomer of each pair is shown. B 2,10-Dihydroxyundecan-6-one forms 2,8-dimethyl-1,7-dioxaspiro[5.5]undecanes (Structure D) JChemEcol and almost absent in synthetic mixtures (Heiduk et al. 2015; Kitching et al. 1989). Spiroacetals constituting 1,6-dioxaspiro[4.5]decanes or 1,6- E1 E2 dioxaspiro[5.5]undecanes produce characteristic mass spectra with a diagnostic doublet forming the most abundant signals. Due to retrocleavage at the six-membered ring, these fragments represent the corresponding 2-methylene-5-alkyl- A2 C1 tetrahydrofuran or 2-methylene-6-alkyltetrahydropyran sub- G2/H1 D1 structures and the corresponding protonated lactone(s). A sec- B1 E4 F2 C2 G1 ond set of key-fragments are produced by α-cleavage of the A1 E3 alkyl substituents adjacent to the ring oxygen (Francke and Relative abundance Kitching 2001). In the present study, those diagnostic signals, the molecular masses of the target compounds, and gas chro- 7891011 12 13 matographic retention times served to classify spiroacetals un- Time (min) ambiguously into different chemical structures, each of which Fig. 2 Representative total ion chromatogram [AT-5 ms column, 40 °C represented a set of stereoisomers that shared unequivocal an- − (1 min hold) to 160 °C at 10 °C/min 1] of headspace volatiles produced alytical data. Chemical structures were termed by letters A-H, by an agitated adult female of Megacyllene caryae. Peak labels indicate and individual isomers within each structure were further des- general chemical structures and elution order, as in Table 1.Spiroacetal ignated by a number indicating the order of elution (e.g., A1 F1 maybeobscuredbyE1. Spiroacetals G3 (12.75) and D2 (10.25), and and A2 had similar mass spectra, with A1 eluting before A2). 1-(methylthio)-2-methylbut-2-ene (5.34) are not present in the headspace

(2E,8E)-isomer D1 and the (2E,8Z)-isomer D2 (Fig. 3D). E, Results with m/z 98, 101, 155, 169, and 184 (M+) was 7-ethyl-2- methyl-1,6-dioxaspiro[4.5]decane. Compound E1, the domi- As expected, headspace samples from males of M. caryae nant component in headspace samples of females of M. caryae contained various components of the aggregation-sex phero- (Figs. 1 and 3E),provedtobethe(7E,2E)-isomer (Table 1), mone, absent in samples from females, including 2- whereas the second most abundant compound, E2, was the phenylethanol, (S)-(−)-limonene, (−)-α-terpineol, neral, and (7E,2Z)-isomer (~9% of E1; Table 1). The remaining two geranial (Online Resource 2). Headspace samples of both compounds, assigned to structure E (E3 and E4,Fig.1)were sexes contained a compound that was tentatively identified present in small amounts (usually <2% of E1; Table 1), and as 1-(methylthio)-2-methylbut-2-ene by a high confidence their (incomplete) spectra suggest the thermodynamically less match of its mass spectrum with that reported in the NIST favored (7Z,2E)- and (7Z,2Z)-stereoisomers, respectively. database, and which may contribute to the characteristic pun- This is corroborated by the fact that the same compounds were gent odor of the agitated beetles. Unexpected, however, was present as trace amounts in the corresponding synthetic sam- the discovery of a suite of spiroacetals in headspace aerations ples of E1 and E2. Structure F with m/z 112, 115, a prominent of females and males of M. caryae. Samples from females fragment at m/z 155, and 184 (M+) was confirmed as 2-ethyl- consistently contained 16 compounds that had mass spectra 7-methyl-1,6-dioxaspiro[4.5]decane (Fig. 3F), and F1 and F2 characteristic of spiroacetals of at least eight distinct chemical were the (2E,7E)- and (2Z,7E)-stereoisomers. Structure G structures (Figs. 2 and 3,Table1). with m/z 98, 101, a diagnostic fragment at m/z 155, and 212 According to its mass spectrum (Francke and Kitching (M+ ), was confirmed as 7-butyl-2-methyl-1,6- 2001) showing a dominant ion doublet at m/z 84, 87, as well dioxaspiro[4.5]decane. The structure of G1,the(7E,2E)-ste- as 156 (M+), structure A (Fig. 3) was assigned as 7-methyl- reoisomer, could be confirmed easily; however, the mass spec- 1,6-dioxaspiro[4.5]decane, represented by its (7E)-isomer, trum of the (7E,2Z)-isomer G2 wasoverlaidbythatofanother A1, and by its less stable (7Z)-isomer, A2 (Table 1). The spiroacetal (Fig. 2, Online Resource 3a). identification of the latter was hampered by an incomplete Due to the low intensity of the target GC peak, the mass spectrum due to its low concentration. Structure B with m/z spectrum obtained from the mixture was incomplete but had 98, 101, and 156 (M+) proved to be 2-methyl-1,6- m/z 169 as the highest detectable signal. The structure of this dioxaspiro[4.5]decane, and B1 was its (2E)-isomer spiroacetal was tentatively assigned after monitoring the diag- (Fig. 3B). Structure C with m/z 98, 101, 155, and 170 (M+) nostic ions at m/z 98, 101, 111, 155, and 169 (SIM; Online was shown to be 2,7-dimethyl-1,6-dioxaspiro[4.5]decane, Resource 3a). The ion pair at m/z 98 and 101 indicated a 2- with C1 as the (2E,7E)-isomer and C2 as the (2Z,7E)-isomer methyltetrahydrofuryl or an unsubstituted tetrahydropyranyl (Fig. 3C). Structure D with m/z 112, 115, 169, and 184 (M+) substructure, whereas the intensity of fragment m/z 111 sug- was 2,8-dimethyl-1,7-dioxaspiro[5.5]undecane with the gested the second ring to be an oxepane (Francke and JChemEcol

Fig. 3 Representative mass spectra of spiroacetal structures recovered from headspace volatiles of agitated adults of Megacyllene caryae and M. robiniae.Figurelabels(A, B, C, etc.) indicate general chemical structures, as in Table 1 (spectra obtained on an HP5973 instrument) JChemEcol

Table 1 Spiroacetals produced by adults of the cerambycid Species and sex (sample size) beetles Megacyllene caryae, M. robiniae,andCallisphyris Structure (isomer) M. caryae M. caryae M. robiniae C. apicicornis apicicornis ♀ (N =10) ♂ (N =10) ♀ (N =5) ♀/♂ (N =13)

BA^: 7-methyl-1,6-dioxaspiro[4.5]decane A1 (7E) 0.03 ± 0.02 (3) A2 (7Z) 0.62 ± 0.61(2) BB^: 2-methyl-1,6-dioxaspiro[4.5]decane B1 (2E)0.24±0.11(5) BC^: 2,7-dimethyl-1,6-dioxaspiro[4.5]decane C1 (2E,7E) 1.73 ± 0.37 (9) 7.81 ± 4.39 (7) 12.0 ± 7.6 (2) C2 (2Z,7E) 0.10 ± 0.05 (4) 6.82 ± 4.3 (2) BD^: 2,8-dimethyl-1,7-dioxaspiro[5.5]undecane D1 (2E,8E) 0.76 ± 0.2 (10) 0.89 ± 0.89 (1) 100 (5) 100 (11) D2 (2E,8Z) <0.01 (1) BE^: 7-ethyl-2-methyl-1,6-dioxaspiro[4.5]decane E1 (7E,2E) 100 (10) 100 (10) E2 (7E,2Z) 9.19 ± 2.4 (10) 12.4 ± 6.4 (4) E3a 0.15 ± 0.06 (5) E4a 1.51 ± 0.32 (9) N/Ab (6) BF^: 2-ethyl-7-methyl-1,6-dioxaspiro[4.5]decane F1 (2E,7E) 69.9 ± 9.1 (5) F2 (2Z,7E) 0.20 ± 0.08 (5) 31.8 ± 14.6 (4) BG^: 7-butyl-2-methyl-1,6-dioxaspiro[4.5]decane G1 (7E,2E) 1.10 ± 0.44 (8) 3.49 ± 2.3 (5) G2c (7E,2Z) 0.06 ± 0.04 (2) G3a 0.02 ± 0.02 (1) BH^: 2-methyl-7-propyl-1,6-dioxaspiro[4.6]undecane H1c (2E,7E) 0.06 ± 0.04 (2)

Compounds are labeled by a letter that indicates the general chemical structure (based on mass spectrum; see text) and a number indicating elution order of stereoisomers from an AT-5 ms column, programmed from 40 °C (1 min hold) to 160 °C at 10 °C/min−1 . The relative configuration is indicated for compounds whose structures were confirmed with authentic standards. Data are expressed as average peak areas (±1 SE) relative to the dominant peak in the chromatograms, per sex and species, while the numbers of headspace samples that contained the compound in detectable quantities are given in parentheses a Relative configuration unknown b Peak area obscured by geranial c Peak area comprises sum of G2 and H1

Kitching 2001). As (7E,2Z)-7-butyl-2-methyl-1,6- Headspace samples from males of M. caryae contained only dioxaspiro[4.5]decane (G2) has a molecular mass (M+)of five of the spiroacetals emitted by conspecific females, but E1 212 and a significant signal at m/z 155 (M+-butyl), the most again was dominant (Table 1). In the present study, however, likely structure of the unknown compound (postulated mass headspace samples from males of M. caryae consistently of 212, but producing a key fragment at m/z 169 [M+-propyl] contained another seven compounds that were not spiroacetals. instead of m/z 155), was either 2-methyl-7-propyl-1,6- Two of these compounds were identified by their mass spectra dioxaspiro[4.6]undecane (H), or 8-propyl-1,7- and comparison with authentic standards as (E)-β-farnesene and dioxaspiro[5.6]dodecane (I). The pattern of of m/z 169 and β-bisabolene. The remaining five compounds were not fully 155 (Online Resource 3a) suggested that the unknown identified, but showed mass spectra indicating oxygenated spiroacetal should elute as a shoulder in front of G2. monoterpenes (U1-U5; Online Resource 4). Stereoisomeric mixtures of H and I were synthesized as ref- Headspace samples from females of M. robiniae contained erences for unambiguous identification (Online Resource 1), and five spiroacetals that matched retention time and mass spectra (2E,7E)-2-methyl-7-propyl-1,6-dioxaspiro[4.6]undecane, (H1) of those identified from female M. caryae, but in very different (Fig. 3H) showed the expected mass spectrum and almost the proportions: D1 was dominant, and F2, E2, C1,andC2 were same GC retention time as G2 (Online Resource 3b). The iso- in much higher relative amounts than in samples from female meric non-natural I, though showing a similar fragmentation M. caryae (Table 1, Fig. 4). A sixth spiroacetal, identified only pattern to H, eluted significantly later; for its mass spectrum, from female M. robiniae, was (2E,7E )-2-ethyl-7-methyl-1,6- seeOnlineResource1. dioxaspiro[4.5]decane, F1. JChemEcol

major components (Bruschini and Cervo 2011; Bruschini et al. 2006; Fortunato et al. 2004; Francke and Kitching 2001). In fact, spiroacetals alone may be sufficient to induce an alarm response by the colony (Dani et al. 2000). Vertebrate predators may learn to associate the odor with a sting, and come to be repelled by the odor alone. In that case, spiroacetals produced by mimics should be the same as those produced by their sympatric models. Consistent with this hypothesis, adult C. apicicornis produce the same spiroacetal as the Neotropical vespid Polybia occidentalis (Olivier): (2E,8E)-2,8-dimethyl-1,7- dioxaspiro[5.5]undecane (Dani et al. 2000), designated D1 in our study. Moreover, the same compound is produced by the vespids Polistes fuscatus (F.) and Eumenes fraternus Say (RFM, Fig. 4 Representative total ion chromatogram of headspace volatiles unpubl. data), and is the primary spiroacetal produced by adults from agitated adults of Megacyllene robiniae (AT-5 ms column, 40 °C of M. robiniae, which often forage side-by-side with these wasps for 1 min., then 10 °C/min−1 to 160 °C) and Callisphyris apicicornis on inflorescences of goldenrod (RFM, unpubl. data). D1 also is −1 (VF5-ms column, 50 °C for 2 min., then 8 °C/min to 270 °C). among the compounds produced by both sexes of M. caryae, Chromatograms trimmed to 6–12 min. (above), or paired with a negative control (below), to improve visibility of peaks. Peak labels which emerge in early spring when overwintering female ves- indicate general chemical structures and elution order, as in Table 1 pids are active. Another potential model of the Megacyllene speciesisthevespidVespula maculifrons (du Buysson), adults of which produce spiroacetals of structures A and B (RFM, Aeration extracts from both sexes of C. apicicornis (Fig. 4) unpub. data). The role of spiroacetals in mimicry is supported contained one spiroacetal: (2E,8E)-2,8-dimethyl-1,7- further by their production by the Palearctic cerambycids dioxaspiro[5.5]undecane, D1 (Fig. 3D, Online Resource 5). Molorchus minor (L.) and Agapanthia villosoviridescens (Meyer 1993), both of which may be visual mimics of Hymenopterans (Linsley 1959; RFM, pers. obs.). In fact, this Discussion hypothesis could explain production of spiroacetals by many other insect species that also appear to be Batesian mimics of Adults of M. caryae and M. robiniae apparently produce aculeate Hymenoptera, including staphylinid beetles, tephritid spiroacetals as a form of chemical defense, because these com- flies, and ichneumonid wasps (Francke and Kitching 2001). pounds are released only when beetles are agitated. This finding The variety of spiroacetals produced by adult Megacyllene explains why spiroacetals were not detected in our earlier work may be a product of selection for chemical mimicry across a on pheromones of the two species (Lacey et al. 2008, broad range of hymenopteran models that differ in their chem- unpublished data). The glandular source of the chemicals has istry of alarm pheromones, and in their geographic distribu- yet to be determined. Males of both species have pores on the tion. In fact, spiroacetals matching the A, B, D, E, and F prothorax that in other species of cerambycines are a source of structures are all produced by various vespid species native pheromones (Ray et al. 2006), but their absence in females to the Palearctic (Francke and Kitching 2001). Alternatively, suggests that they are not the source of spiroacetals. Although the multiple structures of spiroacetals produced by M. caryae Wheeler et al. (1988) did not find spiroacetals in exudate from could be the product of non-selective biosynthesis, which, metasternal glands of agitated adults of either sex of nevertheless, could be adaptive if potential predators are re- M. robiniae, the dominant 11-carbon component, trans- pelled by the appearance and general spiroacetal odor of ves- 2-(trans-1-cis-3-hexadien-1-yl)-5-methyltetrahydrofuran, is pid wasps, rather than by particular isomers of spiroacetals. structurally related to the spiroacetals, suggesting these glands Therefore, we did not specifically look for the enantiomeric may be the source. Metasternal glands of other species of composition of spiroacetals in the cerambycid species we cerambycids produce supposed defensive exudates of different studied. Detailed analyses using enantioselective gas chroma- chemical natures, primarily unsaturated cyclic aldehydes and tography may be necessary to examine special relationships. phenols (reviewed by Millar and Hanks 2016). Spiroacetals also are produced by many insect species that do Production of spiroacetals by adult M. caryae, M. robiniae, not appear to be hymenopteran mimics (Francke and Kitching and C. apicicornis suggests that they mimic their hymenop- 2001), as well as several species of plants (Beck et al. 2008; teran models in odor, as well as in appearance. Social Heiduk et al. 2015; Zhang et al. 2002). They are believed to Hymenoptera recruit nestmates for defense by producing serve as defensive allomones in staphylinid beetles (Huth and alarm pheromones, which commonly contain spiroacetals as Dettner 1990; Zhang et al. 1999), behavioral inhibitors in JChemEcol scolytine bark beetles (Zhang et al. 2002), and sex pheromones Barriga JE, Peña L (1994) New species of Cerambycidae (Coleoptera) – for some species of flies in the genus Bactrocera (Booth et al. from Chile and some synonymies. Gayana Zool 58:91 98 Beck JJ, Higbee BS, Merrill GB, Roitman JN (2008) Comparison of 2009). Thus, spiroacetals of cerambycids may serve purposes volatile emissions from undamaged and mechanically damaged al- other than odor mimicry. For example, the spiroacetal monds. J Sci Food Agric 88:1363–1368 conophthorin (structure A) is produced by some species of trees Berlocher SH, Bouseman JK, McPheron BA, Lyons SA (1992) An elec- and apparently inhibits aggregation in scolytine bark beetles trophoretic study of the red and black morphs of Euderces picipes (Fabricius) (Coleoptera: Cerambycidae). J Kansas Entomol Soc 65: (Francke and Kitching 2001; Zhang et al. 2002), as well as 403–409 disrupting kairomonal attraction of pine sawyers (Monochamus Bezark LG, Monné MA (2013) Checklist of the Oxypeltidae, Vesperidae, species, Cerambycidae) to pheromones of bark beetles Disteniidae and Cerambycidae, (Coleoptera) of the Western (Morewood et al. 2003). The compounds also may be used by Hemisphere. California Department of Agriculture. http://plant. cdfa.ca.gov/byciddb/checklists/WestHemiCerambycidae2013.pdf. Megacyllene species as defensive allomones or alarm phero- Accessed 11 Oct 2016 mones (e.g., Greeney and DeVries 2004), or serve some other Booth YK, Schwartz BD, Fletcher MT, Lambert LK, Kichting W, De intraspecific function. For instance, the fact that spiroacetal E2 Voss JJ (2006) A diverse suite of spiroacetals, including a novel was present at relatively high levels in the blend produced by branched representative, is released by female Bactrocera tryoni – female M. caryae, but was absent in blends produced by males, (Queensland fruit fly). Chem Commun 38:3975 3977 Booth YK, Kitching W, Voss JJD (2009) Biosynthesis of insect suggests that it could serve as a sex-specific signal. spiroacetals. Nat Prod Rep 26:490–525 It also should be noted that the quantitative composition of Bruschini C, Cervo R (2011) Venom volatiles of the paper wasp social a mixture of synthetic stereoisomeric spiroacetals (e.g., E1- parasite Polistes sulcifer elicit intra-colonial aggression on the nest – E4, Fig. 1) likely differs from that of the natural bouquet: the of the host species Polistes dominulus. Insect Soc 58:383 390 Bruschini C, Dani FR, Pieraccini G, Guarna F, Turillazzi S (2006) laboratory synthesis is thermodynamically controlled, where- Volatiles from the venom of five species of paper wasps (Polistes as the biosynthesis of the natural products is enzymatically dominulus, P. gallicus, P. nimphus, P. sulcifer,andP. olivaceus). (kinetically) controlled. As a result, natural blends contain Toxicon 47:812–825 much higher amounts of less stable isomers (e.g., E3 and E4 Cardé RT (2014) Defining attraction and aggregation pheromones: – in Fig. 1) (Francke and Kitching 2001). Additionally, the rel- Teleological versus functional perspectives. J Chem Ecol 40:519 520 Curkovic T, Ferrera C (2012) Female calling and male flight orientation and ative proportion of a mixture will change over time. It is yet searching behaviors in Callisphyris apicicornis: evidence for a female- unknown whether such a time-related change in bouquet is of produced sex attractant pheromone. Cien Inv Agr 39:147–158 any biological significance. Dani FR, Jeanne RL, Clarke SR, Jones GR, Morgan ED, Francke W, In summary, spiroacetals are produced by cerambycid spe- Turillazzi S (2000) Chemical characterization of the alarm phero- mone in the venom of Polybia occidentalis and of volatiles from the cies of diverse taxonomic lineages, and it seems likely that venom of P. sericea. Physiol Entomol 25:363–369 they play an important defensive role in mimicry systems. Deslongchamps P, Rowan DD, Pothier N, Sauvé G, Saunders JK (1981) Moreover, the association of spiroacetals with aculeate 1,7-Dioxaspiro[5.5]undecanes. An excellent system for study of Hymenoptera extends beyond the social wasps (e.g., the potter steroelectronic efforts (anomeric and exo-anomeric effects) in ace- tals. Can J Chem 59:1105–1121 wasp, E. fraternus) and may itself be a Muellerian mimicry Dettner K (1987) Chemosystematics and evolution of beetle chemical system. Our study suggests a rich ecological context for the defenses. Annu Rev Entomol 32:17–48 production and use of spiroacetals, and we hope it serves as a Doubský J, Streinz L, Saman D, Zednik J, Koutek B (2004) foundation for future research on their role in interactions Alkynyltrifluoroborates as versatile tools in organic synthesis: a – between insects and their vertebrate predators. new route to spiroketals. Org Lett 6:4909 4911 Dusham EH (1921) The painted hickory borer, Cyllene caryae,Gahan. Cornell Univ Ag Exp Stn Bull 407:175–203 Eisner T, Schroeder FC, Snyder N, Grant JB, Aneshansley DJ, Utterback Acknowledgments We thank Jocelyn G. Millar for providing a sample D, Meinwald J, Eisner M (2008) Defensive chemistry of lycid bee- of β-bisabolene and assistance with interpreting mass spectra, and tles and of mimetic cerambycid beetles that feed on them. Damon J. Crook for assistance with characterizing isomers of farnesene. Chemoecology 18:109–119 This research was supported by the Alphawood Foundation of Chicago, Fortunato A, Dani FR, Sledge MF, Fondelli L, Turillazzi S (2004) Alarm USDA-NRI grant 2009-35302-05047, and USDA-APHIS grant # 10- communication in Ropalidia social wasps. Insect Soc 51:299–305 8100-1422-CA (to LMH), NIH postdoctoral training grant Francke W, Kitching W (2001) Spiroacetals in insects. Curr Org Chem 5: K12GM000708 (to RFM), the National Commission for Scientific and 233–251 Technological Research-Chile (CONICYT) grant 2007 11070072 (to Francke W, Reith W, Sinnwell V (1980) Bestimmung der relativen TC), and FONDECYT grant 1140779 (to JB). Konfiguration bei Spiroacetalen durch 1H- und 13C-NMR- Spektroskopie (Determination of relative configurations of spiroacetals by 1H- and 13NMR spectroscopy). Chem Ber 113: 2686–2693 References Francke W, Reith W, Bergström G, Tengö J (1981) Pheromone bouquet of the mandibular glands in Andrena haemorrhoa F. (Hym., Barbero F, Thomas JA, Bonelli S, Balletto E, Schönrogge K (2009) Apoidea). Z Naturforsch C 36c:928–932 Queen ants make distinctive sounds that are mimicked by a butterfly Graham EE, Mitchell RF, Reagel PF, Barbour JD, Millar JG, Hanks LM social parasite. Science 323:782–785 (2010) Treating panel traps with a fluoropolymer enhances their JChemEcol

efficiency in capturing cerambycid beetles. J Econ Entomol 103: Perron F, Albizati KF (1989) Chemistry of spiroketals. Chem Rev 89: 641–647 1617–1661 Greeney HF, DeVries PJ (2004) Experimental evidence for alarm phero- Phillips C, Jacobson R, Abrahams B, Williams HJ, Smith L (1980) Useful mones in the neotropical longhorn beetle, Schwarzerion route to 1,6-dioxaspiro[4.4]nonanes and 1,6-dioxaspiro[4.5]decane holochlorum Bates (Coleoptera: Cerambycidae). Coleopt Bull 58: derivatives. J Org Chem 45:1920–1924 642–643 Pothier N, Goldstein S, Deslongchamps P (1992) Cyclization of Handley K, Hough-Goldstein J, Hanks LM, Millar JG, D'Amico V hydroxyenol ethers into spiroacetals. Evidence for the position of (2015) Species richness and phenology of cerambycid beetles in the transition state and its implication on the stereoelectronic effects urban forest fragments of northern Delaware. Ann Entomol Soc in acetal formation. Helv Chim Acta 75:604–620 Am 108:251–262 Raske AG (1967) Morphological and behavioral mimicry among beetles Heiduk A, Kong H, Brake I, von Tschirnhaus M, Tolasch T, Tröger AG, of genus Moneilema. Pan-Pac Entomol 43:239–244 Wittenberg E, Francke W, Meve U, Dötterl S (2015) Deceptive Ray AM, Lacey ES, Hanks LM (2006) Predicted taxonomic patterns in Ceropegia dolichophylla fools its kleptoparasitic fly pollinators with pheromone production by longhorned beetles. Naturwissenschaften exceptional floral scent. Front Ecol Evol 3:66 93:543–550 Huth A, Dettner K (1990) Defense chemicals from abdominal glands of Ruxton GD, Sherratt TN, Speed MP (2004) Avoiding attack: The evolu- 13 rove beetle species of subtribe Staphylinina (Coleoptera: – tionary ecology of crypsis, warning signals and mimicry. Oxford Staphylinidae, Staphylininae). J Chem Ecol 16:2691 2711 University Press, Oxford Jacobson R, Taylor RJ, Williams HJ, Smith LR (1982) Naturally occur- Slobodchikoff CN (1987) Aversive conditioning in a model-mimic sys- ring spirocyclic ketals from lactones. J Org Chem 47:3140–3142 tem. Anim Behav 35:75–80 Kitching W, Lewis JA, Perkins MV, Drew R, Moore CJ, Schurig V, Š König WA, Francke W (1989) Chemistry of fruit flies. vácha P, Lawrence JF (2014) Chapter 2.4 Cerambycidae Latreille, Composition of the rectal gland secretion of (male) Dacus cucumis 1802. In: Leschen RAB, Beutel RG (eds) Handbook of zoology: (cucumber fly) and Dacus halfordiae. Characterization of (Z,Z)-2,8- Arthropoda: Insecta: Coleoptera, beetles, Morphology and system- – atics (Phytophaga), vol 3. Walter de Gruyter, Berlin/Boston, pp. dimethyl-1,7-dioxaspiro[5.5]undecane. J Org Chem 54:3893 3902 – Lacey ES, Moreira JA, Millar JG, Hanks LM (2008) A male-produced 77 177 aggregation pheromone blend consisting of alkanediols, terpenoids, Vereecken NJ, McNeil JN (2010) Cheaters and liars: Chemical mimicry – and an aromatic alcohol from the cerambycid beetle Megacyllene at its finest. Can J Zool 88:725 752 caryae. J Chem Ecol 34:408–417 Waldbauer GP (1988) Asynchrony between Batesian mimics and their – Linsley EG (1959) Mimetic form and coloration in the Cerambycidae models. Am Nat 131:S103 S121 (Coleoptera). Ann Entomol Soc Am 52:125–131 Wheeler JW, Abraham M, Highet RJ, Duffield RM (1988) Metasternal Meyer H (1993) Identifizierung und Synthese flüchtiger Inhaltsstoffe gland secretion of the locust tree borer, Megacyllene robiniae holzschädigender Käfer. PhD dissertation, University of Hamburg, (Coleoptera: Cerambycidae). Comp Biochem Physiol B 91:771– Germany 775 Millar JG, Hanks LM (2016) Chemical ecology of cerambycid beetles. Zhang H, Fletcher MT, Dettner K, Francke W, Kitching W (1999) In: Wang Q (ed) Cerambycidae of the world: Biology and manage- Synthesis and absolute stereochemistry of spiroacetals in rove bee- ment. CRC Press/Taylor & Francis, Boca Raton (in press) tles (Coleoptera: Staphylinidae). Tetrahedron Lett 40:7851–7854 Mitchell RF (2012) Chemical communication in cerambycid beetles and Zhang Q-H, Tolasch T, Schlyter F, Francke W (2002) Enantiospecific the molecular basis of olfaction. PhD Dissertation, University of antennal response of bark beetles to spiroacetal (E)-conophthorin. Illinois at Urbana-Champaign, Urbana, IL J Chem Ecol 28:1839–1852 Mitchell RF, Hughes DT, Luetje CW, Millar JG, Soriano-Agatón F, Hanks LM, Robertson HM (2012) Sequencing and characterizing odorant receptors of the cerambycid beetle Megacyllene caryae. Insect Biochem Mol Biol 42:499–505 Dedication Morewood WD, Simmonds KE, Gries R, Allison JD, Borden JH (2003) Disruption by conophthorin of the kairomonal response sawyer bee- Dedicated to Prof. Dr. G. Bringdmann, on the occasion of his 65th tles to bark beetle pheromones. J Chem Ecol 29:2115–2129 birthday.